Porous semiconductor-based optical interferometric sensor

ABSTRACT

The measurement of the wavelength shifts in the reflectometric interference spectra of a porous semiconductor substrate such as silicon, make possible the highly sensitive detection, identification and quantification of small analyte molecules. The sensor of the subject invention is effective in detecting multiple layers of biomolecular interactions, termed “cascade sensing”, including sensitive detection of small molecule recognition events that take place relatively far from the semiconductor surface.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of application Ser. No. 08/924,601, filedSep. 5, 1997, now abandoned whose disclosures are incorporated herein byreference.

GOVERNMENTAL SUPPORT

This invention was made with governmental support under Contract No.N00014-95-1-1293 by the ONR. The government has certain rights in theinvention.

TECHNICAL FIELD

This invention is related to solid state sensors and, more particularly,to the use and preparation of a porous semiconductor such as a siliconwafer for the quantitative and qualitative analysis of an analyte suchas an organic analyte.

BACKGROUND OF THE INVENTION

Solid-state sensors and particularly biosensors have receivedconsiderable attention lately due to their increasing utility inchemical, biological, and pharmaceutical research as well as diseasediagnostics. In general, biosensors consist of two components: a highlyspecific recognition element and a transducing structure that convertsthe molecular recognition event into a quantifiable signal. Biosensorshave been developed to detect a variety of biomolecular complexesincluding oligonucleotide pairs, antibody-antigen, hormone-receptor,enzyme-substrate and lectin-glycoprotein interactions. Signaltransductions are generally accomplished with electrochemical,field-effect transistor, optical absorption, fluorescence orinterferometric devices.

It is known that the intensity of the visible photoluminescence changesof a porous silicon film depend on the types of gases adsorbed to itssurface. Based on this phenomenon, a simple and inexpensive chemicalsensor device was developed and disclosed in U.S. Pat. No. 5,338,415.

As disclosed in that patent, porous films of porous films of silicon(Si) can be fabricated that display well-resolved Fabry-Perot fringes intheir optical reflectance properties. The production of a porous silicon(Si) layer that is optically uniform enough to exhibit these propertiesmay be important for the design of etalons (thin film opticalinterference devices for laser spectroscopy applications) and otheroptical components utilizing porous Si wafers. Such interference-basedspectra are sensitive to gases or liquids adsorbed to the inner surfacesof the porous Si layer.

Ever increasing attention is being paid to detection and analysis of lowconcentrations of analytes in various biologic and organic environments.Qualitative analysis of such analytes is generally limited to the higherconcentration levels, whereas quantitative analysis usually requireslabeling with a radioisotope or fluorescent reagent. Such procedures aretime consuming and inconvenient. Thus, it would be extremely beneficialto have a quick and simple means of qualitatively and quantitativelydetect analytes at low concentration levels. The invention describedhereinafter provides one such means.

BRIEF SUMMARY OF THE INVENTION

The subject invention contemplates the detection and, if desired,measurement of the wavelength shifts in the reflectometric interferencespectra of a porous semiconductor substrate such as a silicon substratethat make possible the highly sensitive detection, identification andquantification of small molecules and particularly, small organicmolecules (i.e., carbon-containing molecules e.g., biotin, and thesteroid digoxigenin), short DNA oligonucleotides (e.g., 16-mers), andproteins (e.g., streptavidin and antibodies). The binding of inorganicspecies such as metal ions is also contemplated. Most notably, thesensor of the subject invention has been shown to be highly effective indetecting multiple layers of biomolecular interactions, termed “cascadesensing”, including sensitive detection of small molecule recognitionevents that take place relatively far from the silicon surface.

In an exemplary embodiment, a p-type silicon (Si) wafer (substrate) isgalvanostatically etched in a hydrofluoric acid (HF)-containingsolution. The etched wafer is rinsed with ethanol and dried under astream of nitrogen gas. Reflection of white light off the porous siliconresults in an interference pattern that is related to the effectiveoptical thickness. The binding of an analyte to a recognition partnerimmobilized in the porous silicon substrate results in a change in therefractive index, which is detected as a wavelength shift in thereflection interference pattern.

One benefit of the present invention is the provision of a device fordetecting the presence of target (analyte) molecules such as biologicalor organic compound molecules at very low concentrations.

An advantage of the present invention is the provision of a means fordetecting the presence of multilayered molecular assemblies.

Still another benefit of the present invention is a device that iscapable of quantitatively detecting an analyte.

Still another advantage of the present invention is that the presence ofan analyte in a sample solution can often be detected by visualinspection, and without the need for special apparatus.

Still further benefits and advantages will be apparent to a worker ofordinary skill from the disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the present invention will be facilitated byconsideration of the following detailed description of a preferredembodiment of the present invention, taken in conjunction with theaccompanying drawings, in which like reference numerals refer to likeparts and in which:

FIG. 1 is a schematic representation of the porous semiconductor, e.g.,silicon-based optical interferometric biosensor of the presentinvention.

FIGS. 2A and 2B are interferometric reflectance spectra of DNA-modifiedporous Si layers.

FIG. 3 shows the change in effective optical thickness in aDNA-A-modified porous Si layer as a function of DNA-A' concentration.

FIGS. 4A, 4B, 4C and 4D are cascade sensing and reflectometricinterference spectra of multilayered molecular assemblies.

DETAILED DESCRIPTION OF THE INVENTION

A contemplated interferometric sensor is depicted in FIG. 1 and isextremely sensitive in detecting the presence of a number of ligands(analytes) that bind specifically to a chemical binder on the sensorsurface. For example, the lowest DNA concentration measured with acontemplated porous Si interferometric sensor was 9 fg/mm². Forcomparison, the detection limits of current technologies are:Interferometry (100 pg/mm²); Grating Couplers (2.5 pg/mm²); SurfacePlasmon Resonance (10 pg/mm²).

The devices and methods of the present invention employ a poroussemiconductor layer as an element of their interferometric sensors. A“porous semiconductor layer” is a porous layer having a relativelyconsistent thickness, relatively consistent porosity and made up of asemiconducting solid that is relatively transparent. A “semiconducting”material is one having a bulk resistivity of from about 1 to about 1×10⁷ohms per cm.

The term “transparent” as used herein refers to the property of amaterial to transmit a fraction, such as at least about 20% of asuitable range of wave lengths of light from which Fabry-Perot fringescan be generated.

The term “light” is employed herein to include not only the visibleportion of the electromagnetic spectrum, i.e. 350-800 nm, but also theinfrared region of from say 800-3000 nm and the ultraviolet region offrom about 50-350 nm. Longer and shorter wavelengths can be employed aswell. The wavelengths employed can play a part in the selection of layerthickness and pore size of the porous semiconductor layer. As a generalrule shorter wavelengths permit thinner layer thicknesses and smallerpore sizes while longer wavelengths permit thicker layer thicknesses andlarger pose sizes.

The porous semiconductor layer can range in thickness from about 0.5 toabout 30 microns with thicknesses of from about 1 or 2 to about 10microns being preferred when visible light such as white light isemployed and with thicknesses of from about 5 to about 30 microns beingpreferred with infrared wave lengths and thicknesses of from about 0.5to 5 microns being preferred with ultraviolet wave lengths.

The pores (or cavities) in the porous semiconductor layers are typicallysized in terms of their nominal “diameter” notwithstanding the fact thatthey are somewhat irregular in shape. These diameters range from about 2nm to about 2000 nm with diameters of from about 10 to about 200 nmbeing preferred for visible light and 2-50 nm diameters being preferredfor ultraviolet light and 100 to 2000 nm being preferred for infraredlight. The surface of the solid semiconductor is flat with a substantialdegree of porosity such as from about 10% to about 80% of the surfacearea and typically from 20 to 70% of the surface area.

The semiconducting porous layer can be formed of any semiconductorcapable of being formed into the porous structure of the desiredthickness and porosity. Silicon and silicon alloys are preferredsemiconductors because of their amenability to the preferred galvanicetching process described herein for forming porous structures. Thesematerials can include p-doped silicon, n-doped silicon, intrinsic(undoped) silicon, as well as alloys of these materials with, forexample germanin in amounts of up to about 10% by weight as well asmixtures of these materials.

A representative device depicted in FIG. 1 is prepared from anelectrochemical etch of a semiconductor such as single-crystal p-type(boron-doped) silicon wafers that produce microporous silicon thatdisplays well-resolved Fabry-Perot fringes in its reflectometricinterference spectrum. Silicon-containing (silicious) semiconductors arepreferred herein, and although p-type silicon wafers are utilized hereinas exemplary substrates, it is to be understood that n-type silicon andundoped, intrinsic silicon can be used, as a silicon-germanium (Si-Ge)alloy containing up to about 10 mole percent germanium, Group IIIelement nitrides and other etchable semiconductor substrates. Exemplarysemiconductor substrates and dopants are noted below.

n dopant p dopant H₂Se (CH₃)₂Zn H₂S (C₂H₅)₂ Zn (CH₃)₃Sn (C₂H₅)₂ Be(C₂H₅)₃Sn (CH₃)₂Cd SiH₄ (ηC₂H₅)₂Mg Si₂H₆ B P Al As Ga Sb In

The substrate can be GaAs, Si, Al₂O₃, MgO, TiO₂, SiC, ZnO, LiGaO₂,LiAlO₂, MgAl₂O₄ or GaN.

Reflection of light at the top (surface) and bottom of the exemplaryporous semiconductor layer results in an interference pattern that isrelated to the effective optical thickness (product of thickness L andrefractive index n) of the film by eq. 1,

mλ=2 nL   (1)

where m is the spectral order and μ is the wavelength of light. Bindingof an analyte to its corresponding recognition partner, immobilized onthe porous silicon substrate area results in a change in refractiveindex of the layer medium and is detected as a corresponding shift inthe interference pattern.

The refractive index, n, for the porous semiconductor in use is relatedto the index of the semiconductor and the index of the materials present(contents) in the pores pursuant to eq. 2

n=(1-P)n_(semiconductor)+Pn_(contents)   (2)

Where P=porosity of porous semiconductor layer;n_(semiconductor)=refractive index of semiconductor;n_(contents)=refractive index of the contents of the pores.

The index of refraction of the contents of the pores changes when theconcentration of analyte species in the pores changes. Most commonly,the analyte (target) species is an organic species that has a refractiveindex that is larger than that of the semiconductor. The replacement ofa species of lower index of refraction (water) by another species ofhigher index of refraction (analyte) would be expected to lead to anincrease in the overall value for index of refraction. An increase inindex should result in a shift in the interference pattern wavelengthsto longer values; i.e., a bathochromic or “red” shift pursuant toequation 1. Contrarily, the observed shift in interference patternwavelengths is opposite that which is expected; i.e., is toward shorterwavelengths exhibiting a hypsochromic or “blue” shift.

The basis for the observed wavelength blue shift is not understood withcertainty. However, the observed, unexpected hypsochromic shift inwavelengths is believed to be the result of a reduction in the index ofrefraction of the semiconductor itself that is induced by the intimateassociation of the semiconductor with the bound analyte.

White light is preferred for carrying out reflectance measurements, andis used illustratively herein. The use of white light or other light inthe visual spectrum can permit a determination of the presence of ananalyte in a sample by visual inspection of a color change in thereflected light without the need of special apparatus. It should beunderstood, however, that reflected infrared (IR) and ultraviolet (UV)light canals be utilized along with an appropriate spectral measuringdevice.

The sensors of the present invention include the binder molecule (alsoreferred to as the “recognition partner”) for the analyte and the likethat is bound to or otherwise intimately associated with the poroussemiconductor surface. This intimate association can be accomplished byany approach that leads to the tethering of the binder molecule to thesemiconductor. This includes without limitation covalently bonding thebinder molecule to the semiconductor, ionically associating the bindermolecule to the substrate, adsorbing the binder molecule onto thesurface of the semiconductor, or the like. Such association can alsoinclude covalently attaching the binder molecule to another moiety,which in turn is covalently bonded to the semiconductor, bonding thetarget molecule via hybridization or another biological associationmechanism to another moiety with is coupled to the semiconductor.

The binding of an analyte to its corresponding recognition partner,immobilized on the porous silicon substrate, results in a change inrefractive index of the layer medium and is detected as a correspondingshift in the interference pattern. Recognition partners or bindingcompounds can be peptides, small molecules (molecular weight of lessthan about 500), metal ions and their preferably organic bindingligands, antibodies, antigens, DNA, RNA or enzymes. More broadly, arecognition partner can be any receptor of an acceptor molecule that canbe adsorbed by the substrate and binds to a ligand provided by ofanother molecule or ion.

More specifically, the Examples that follow illustrate use of twodifferent single strands of binder DNA (SEQ ID NOs:1 and 2) bound to theporous silicon substrate, and two different single DNA strands (SEQ IDNOs:3 and 4, respectively) as analyte (Examples 1 and 3). Example 4illustrates the use of a biotin-bound porous silicon substrate withstrepavidin, as well as biotnylated anti-mouse antibodies that were usedto analyze for mouse-anti-digoxigenin, and those antibodies were thenused to assay for the presence of digoxigenin. Further exemplary bindingpairs include so-called polypeptide P-62 (SEQ ID NO:5) of U.S. Re.33,897 (1992), whose disclosures are incorporated by reference, withhuman antibodies to the Epstein-Barr nuclear antigen (EBNA) as analyte;monoclonal antibodies ATCC HB 8742 or HB 8746 that immunoreact withhuman apolipoprotein B-100 as analyte, or monoclonal antibodies ATCC HB9200 or HB 9201 that immunoreact with human apolipoprotein A-I asanalyte as are described in U.S. Pat. No. 4,828,986, whose disclosuresare incorporated by reference; and the several deposited monoclonalantibodies listed at column 13 of U.S. Pat. No. 5,281,710, and theirlisted binding partners as analyte, which disclosures are incorporatedby reference.

Electrochemical etching of Si can generate a thin (approximately 1-10μm) layer of porous Si on the silicon substrate with cavities of about10 nm to about 200 nm in diameter, providing a large surface area forbiomolecular interaction inside the porous Si layer. The porous filmsare uniform and sufficiently transparent to display Fabry-Perot fringesin their optical reflection spectrum.

More particularly, a porous Si substrate is prepared by anelectrochemical etch of a polished (100)-oriented p-type silicon(B-doped 3 Ohm-cm resistivity) wafer. The etching solution is preparedby adding an equal volume of pure ethanol to an aqueous solution of HF(48% by weight). The etching cell is constructed of Teflon® and is opento air.

Si wafers are cut into squares with a diamond scribe and mounted in thebottom of the Teflon® cell with an O-ring seal, exposing 0.3 cm² of theSi surface. Electrical contact is made to the back side of the Si waferwith a strip of heavy aluminum foil, such as heavy duty householdaluminum foil. A loop of platinum wire is used as a counter-electrode.The exposed Si face can be illuminated with light from a tungsten lampfor the duration of the etch in order to enhance the optical propertiesof the films. Etching is illustratively carried out as a 2-electrodegalvanostatic operation at an anodic current density of 5 mA/cm² for 33minutes. After etching, the samples are rinsed in ethanol and driedunder a stream of N₂. Scanning electron microscopy and atomic forcemicroscopy showed that porous silicon films so prepared were about 5-10microns thick and contained an average of 200 nm diameter pores.

The porous semiconductor so prepared was modified by oxidation withbromine gas in an evacuated chamber for one hour, followed by hydrolysisin air. The molecular recognition elements were then attached to theresulting silicon dioxide surface using conventional techniques.

The sensors of this invention can be employed as discrete, independentunits. Multiple sensors can also be arrayed together. Where multiplesensors are desired to be arrayed together, a plurality of porous areascan be etched on to the surface of a single semiconductor substrate inmuch the same way as microchip patterns are prepared A plurality ofseparate porous areas can also be combined to form a desired array.

An array of sensors can make it possible to have a plurality ofconcentrations of a single binder molecule on a single plate so as toprovide a “dose-response curve” for a particular analyte. Multiplesensors also can make it possible to have a plurality of differentbinder molecules on the same plate so as to make multiple screenings ina single test.

A sensor having a plurality of individual porous areas can be analogizedto a multi-well microtiter plate, and can contain the same or differentassociated binder compound at any desired porous area so that the sameor a different binding assay can be carried out on each porous area. Theindividual binder compound-porous areas are then illuminated. Bindingstudies with analytes are then carried out for those areas, followed byreillumination. Binding results are obtained in a manner similar to thatused for the individual porous areas exemplified herein.

Spectral Measurement. To measure optical interference spectra, aPrinceton Instruments CCD photodetector/Acton research 0.25 mmonochrometer, fitted with a fiber optic and microscope objective lensto permit detection from small (<1 mm²) sample areas was used for thestudies described here, but similar equipment is well-known and can beused instead. The white light source for the experiments was a lowintensity krypton, tungston or other incandescent bulb. A linearpolarizing filter was used to enhance the appearance of the interferencespectra.

The substrate can be pre-treated with a chemical receptor (bindercompound) species (such as an antibody) to provide chemical specificity.For gas measurements, the sample was mounted in a Pyrex® dosing chamberand exposed to the gaseous analyte of interest. For liquid-phasemeasurements, as in an aqueous medium, a Teflon® and O-ring cell similarto the cell employed in etching the porous layer was used. Measurementshave also been taken using a liquid flow-through chamber equipped withglass or plastic window.

The fringe pattern can be changed by replacing the air or liquid in thepores with a material of differing refractive index. The shift in fringemaxima corresponds to a change in the average refractive index of thethin film medium. Solution of the simultaneous equations provided bymeasurement of the fringe spacing provides a quantitative measurementthat can be related to the analyte concentration. Chemical specificitycan be introduced by incorporating or chemically bonding molecularrecognition agents such as peptides, antibodies, antigens, single- ordouble-strand DNA or RNA, enzymes, a metal ion-binding ligand and thelike onto the inner surfaces of the porous Si film. Control measurementscan be performed on a similar sample that does not contain the molecularrecognition elements. Further details as to the preparation of a poroussilicon substrate and apparatus used for spectral measurements can befound in U.S. Pat. No. 5,338,415, whose disclosures are incorporated byreference.

Thus, one aspect of the invention contemplates a process for detecting aanalyte molecule such as an organic molecule analyte. In accordance withthat process, a porous silicon substrate is provided and prepared, andthat prepared substrate is provided and contacted with a binder compoundto form a binder compound-bound substrate. The wavelength maximum of theFabry-Perot fringes is determined upon illumination of the bindercompound-bound substrate. That binder compound-bound substrate isthereafter contacted with a sample to be assayed that may contain ananalyte that is an organic molecule that binds to the binder compound ofthe substrate. When the desired analyte is present in the sample, indistilled water or various buffer solutions that ligand binds to thebinder compound to form a ligand-bound substrate. The contact betweenthe sample and binder compound-bound substrate can be maintained for afew seconds to several hours, as desired to form the ligand-boundsubstrate. When the substrate is thereafter reilluminated with the samelight source, a shift in the wavelength maximum of the Fabry-Perotfringes from that previously determined indicates the detection andtherefore presence of the analyte in the sample.

Without committing to any particular theory in support of the subjectinvention, it is believed that the unique sensitivity of the systeminvolves selective incorporation or concentration of an analyte such asan illustrative organic analyte in the porous Si layer to modify therefractive index by two effects: increase of the average refractiveindex of the medium in the pores by replacing water (refractive index1.33) with organic matter (refractive index typically 1.45), and alsodecrease of the refractive index of the Si by modifying the carrierconcentration in the semiconductor. A net increase in refractive indexis expected to shift the interference spectrum to longer wavelengths,whereas a decrease in index is expected to shift the spectrum to shorterwavelengths. Without exception, a shift to shorter wavelengths in suchcases has been observed, indicating that the induced change in thesemiconductor overwhelms the refractive index change occurring in thesolution phase.

Each of Examples 1-3 was carried out in 1.0 M aqueous NaCl at 25° C.,whereas Examples 4 and 5 were carried out in 0.5 M NaCl.

EXAMPLE 1

Binder DNA oligonucleotide-derivatized porous silicon films wereemployed to test the selectivity and limits of detection of acontemplated sensor. For attachment of DNA, atrimethoxy-3-bromoacetamido-propylsilane linker was synthesized byreaction of bromoacetic acid with trimethoxy-(3-aminopropyl)silane inthe presence of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride in methylenechloride as solvent. The linker product was purified by columnchromatography on silica gel. The oxidized porous silicon samples werethen contacted with a toluene solution of the linker for 2 hours. Theresulting linker-bound substrate was thoroughly rinsed with pure tolueneand methylene chloride, and dried for about 18 hours under reducedpressure.

HPLC-Purified 5′-phopsphorothiate oligonucleotides (DNA-A and DNA-B,illustrated hereinafter) were separately dissolved at about 50 nmol in asolution of 1:1:0.2; water/DMF/5%NaHCO₃ and admixed with thelinker-bound porous semiconductor substrate for about 2 hours. Thepresence of the DNA-modification on the porous surface of the resultingbinder compound-bound substrate was confirmed by FTIR spectroscopy.

In the presence of complementary analyte DNA sequences (DNAconcentrations ranging from 2×10⁻¹⁵ M to 2×10⁻⁶ M) pronounced wavelengthshifts in the interference pattern of the porous silicon films wereobserved (FIG. 2). Under similar conditions but in the presence ofnoncomplementary DNA sequences, no significant shift in the wavelengthof the interference fringe pattern was detected-only minor amplitudefluctuations were observed.

Specifically, measurements were made of two DNA sequences:

DNA-A: 5′-pGC CAG AAC CCA GTA GT-3′ SEQ ID NO:1

and

DNA-B: 5′-CCG GAC AGA AGC AGA A-3′ SEQ ID NO:2,

and corresponding complementary strands [(DNA-A' (SEQ ID NO:3) andDNA-B'v (SEQ ID NO:4)]. For clarity, only one set of data are shown.

In FIG. 2A, the Fabry-Perot fringes 10 from a porous Si surfacederivatized with DNA-A are shown to shift to shorter wavelength 11 uponexposure to a 2×10⁻¹² M solution of DNA-A' (the complementary sequenceof DNA-A) in 1 M NaCl (aq). The net change in effective opticalthickness (from 7,986 to 7,925 nm) upon DNA-A' recognition isrepresented by the difference 12 between the two interference spectra.

EXAMPLE 2

FIG. 2B represents a control for Example 1, showing the Fabry-Perotfringes 10 a of a DNA-A derivatized porous Si surface before and afterexposure to a 2×10⁻¹² M solution of DNA-B (non-complementary sequence)in 1 M NaCl (aq). No wavelength shift was observed up to the measuredconcentration of 10⁻⁹ M of DNA-B.

EXAMPLE 3

Fluorescence spectroscopy was used to independently investigate thesurface coverage of immobilized DNA on porous Si and the rate of analytediffusion into the Si substrate for the purposes of comparison with thesubject invention. Solutions of fluorescein-labeled analytecomplementary DNA oligonucleotides were placed in fluorescence cuvettesand the binder DNA-derivatized porous Si substrate was then added to thecell without stirring. At lowest DNA concentrations employed in thestudy, the fluorescence intensity of the samples decreased to anasymptotic limit in 40 min (similar equilibration times were observed inthe interferometric measurements described above) (FIG. 3). The dataindicate 1.1×10⁻¹² mol of bound DNA in a 1 mm² porous Si substrate(calculated from standardized fluorescence titration curves). The dataobtained from the reflectometric interference measurements also provideda similar coverage number.

EXAMPLE 4

The subject invention was used to sense multiple layers of biomolecularinteractions (cascade sensing) and small molecule detection. A linkerwith attached biotin was prepared by reaction of Iodoacetyl-LC-biotin(Pierce Biochemicals) with 3-mercaptopropyl-trimethoxysilane (AldrichChemicals) in dimethylformamide (DMF). After purification, thebiotinylated linker was dissolved in ethanol or DMF and the oxidized,porous semiconductor was immersed in the solution for 12 hours. Thesample was then rinsed thoroughly with ethanol, and dried under a streamof nitrogen to provide a binder compound-bound substrate.

Exposure of a biotinylated (binder) porous Si substrate to a 5×10⁷ Manalyte streptavidin solution resulted in a large blue-shift of theinterference fringes, corresponding to a decrease in the measuredeffective optical thickness from 12,507-11,994 nm (the loweststreptavidin concentration employed was 10⁻¹⁴ M) (FIG. 4A). Controlstudies performed by exposing a biotinylated porous Si substrate toinactivated streptavidin (streptavidin pre-saturated with biotin) didnot display perceptible shifts in interference pattern.

The biotin-streptavidin monolayer surface was contacted with aqueous10⁻⁸ M biotinylated anti-mouse IgG (from goat IgG). Binding of thissecondary antibody to the surface was indicated by a decrease ineffective optical thickness of the monolayer from 11,997 to 11,767(lowest concentration employed with a detectable signal was 10⁻¹² M)(FIG. 4B). Treatment of the secondary antibody sample withanti-digoxigenin (mouse IgG) at a concentration of 10⁻⁸ M caused afurther decrease in the effective optical thickness of the monolayerfrom 11,706 to 11,525 nm (FIG. 4C). The interaction of digoxigenin (10⁻⁶M), a steroid with molecular weight of 392, with the anti-digoxigeninIgG-bound porous Si surface was also detected with a decrease of theeffective optical thickness from 11,508 to 11,346 nm (FIG. 4D).

EXAMPLE 5

To rule out the possibility of nonspecific interaction, anon-biotinylated surface was subjected to the same solution, andconditions as described in Example 4. No measurable change in theeffective optical thickness was observed on treatment with streptavidin,secondary antibody, primary antibody, and digoxigenin. Detection of therelatively small biotin molecule (MW=244) at concentrations as low as10⁻¹² M has also been demonstrated using biotin-streptavidin-modifiedporous Si.

Although the invention has been described with reference to a preferredembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments and equivalents falling within the scope ofthe appended claims.

Various features of the invention are set forth in the following claims.

5 1 16 DNA Artificial Sequence Description of Artificial Sequencesynthetic DNA sequence 1 gccagaaccc agtagt 16 2 16 DNA ArtificialSequence Description of Artificial Sequence synthetic DNA sequence 2ccggacagaa gcagaa 16 3 16 DNA Artificial Sequence Description ofArtificial Sequence synthetic DNA sequence 3 actactgggt tctggc 16 4 16DNA Artificial Sequence Description of Artificial Sequence synthetic DNAsequence 4 ttctgcttct gtccgg 16 5 20 PRT Artificial Sequence Descriptionof Artificial Sequence synthetic polypeptide related to Epstein-BarrVirus Nuclear Antigen 5 Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly AlaGly Gly Gly Ala 1 5 10 15 Gly Gly Ala Gly 20

What is claimed is:
 1. Means for detecting shifts in Fabry-Perot fringeswith a solid state sensor, said fringes being reflected from asubstrate, said means for detecting comprising a semiconductor substratehaving a porous surface, said substrate surface further having aplurality of discrete and separate regions, and having an organic bindercompound adsorbed thereon, said substrate having a thickness selected togenerate Fabry-Perot fringes from reflection of light therefrom.
 2. Thedetecting means of claim 1 further including an analyte that binds tosaid organic binder compound, said semiconductor substrate, whencontaining said analyte bound to said organic binder compound reflectinglight to exhibit Fabry-Perot fringe wavelengths different from thoseexhibited when the analyte is not so bound.
 3. A reflective sensor foran analyte having a layer of porous semiconductor with a plurality ofdiscrete and separate sites having one or more binder compounds specificto the analyte, said layer being substantially transparent and having atop surface and a bottom surface which reflect light to exhibitFabry-Perot fringes having a first set of characteristic wavelengths inthe absence of analyte and second set of characteristic wavelengths whenanalyte is present, said second set of characteristic wavelengths beingdetectably shifted from said first set of characteristic wavelengths. 4.The detecting means of claim 3 wherein said layer of poroussemiconductor with a binder compound specific to the analyte exhibits afirst index of refraction, wherein said analyte exhibits a second indexof refraction which is greater than said first index of refraction, butwherein the second set of characteristic wavelengths is shorter than thefirst set of characteristic wave lengths.
 5. The detecting means ofclaim 4 wherein said semiconductor comprises silicon.
 6. A means fordetecting shifts in Fabry-Perot fringes for use in detecting a targetspecies comprising a porous semiconductor layer with a plurality ofdiscrete and separate regions having one or more binder compounds, of athickness selected to generate Fabry-Perot fringes from the reflectionof light therefrom, a binding material specific to the target species,said Fabry-Perot fringes having a first set of characteristics peakwavelengths in the absence of the target species and a second set ofcharacteristic peak wavelengths in the presence of the target specieswith the second, set of peak wavelengths being shifted toward shorterwavelengths relative to said first set of wavelengths.
 7. The detectingmeans of claim 6 wherein said semiconductor comprises silicon.
 8. Thedetecting means of claim 7 wherein the porous silicon has a first indexof refraction and said target species has a second index of refractionwhich is higher than said first index of refraction.
 9. The detectingmeans of claim 8 additionally comprising a binder material specific tothe porous silicon layer, said binder material specifically binding thetarget species.
 10. A means for detecting a shift in Fabry-Perotfringes, including a reflective sensor array for at least one analyte,said array comprising a layer of porous semiconductor with a pluralityof discrete and separate regions having one or more binder compoundsspecific to at least one of the at least one analyte, said layer beingsubstantially transparent and having a top surface and a bottom surfacewhich reflect light in each of the plurality of regions to exhibitFabry-Perot fringes for such regions having a first set ofcharacteristic wave lengths in the absence of the at least one analyteand a second set of characteristic wave lengths when analyte is present,said second set of characteristic wave lengths being detectably shiftedfrom said first set of characteristic wavelengths.